PROJECT SUMMARY Prions cause rapidly progressive neurodegenerative diseases clinically characterized by cognitive impairment, cerebellar ataxia, and death within approximately seven months in the case of sporadic Creutzfeldt-Jakob disease. Prion disorders occur following the auto-catalytic conversion and aggregation of the cellular prion protein. The structural determinants of the prion protein (PrP) that govern prion conversion and enable pathogenic prions to spread into and through the CNS, targeting distinct cell populations, are unknown. A major goal of this proposal is to understand the mechanisms that underlie prion conversion and cell-specific targeting. In this project, we have previously pursued a broad range of approaches, from in vitro conversion assays to newly generated transgenic mice, to determine the role of a PrP segment having exceptionally high sequence variability among mammals, the ?2-?2 loop (residues 165-175). We discovered that the ?2-?2 loop is a key segment of cellular PrP that impacts cross-species prion transmission, impedes transmission of deer and elk prions to humans, and alters the pathogenic prion conformation in a sequence-dependent manner. We also found that amino acid substitutions in the loop led to de novo prion disease in mice. Finally, we determined that elements of the PrP primary sequence, and not secondary structure, control prion conversion. In this renewal, we aim to identify the key contributors to prion aggregate assembly. We build on our observation that structural features of PrP together with host glycosaminoglycans drive efficient prion conversion. First, we will identify the highly amyloidogenic interacting segments that trigger prion conversion and will test rationally-designed inhibitors to block aggregation. Second, we will define how post-translational modifications (PTM) of PrP modify the prion plaque morphology and pathogenesis using mouse models expressing PTM variants of PrP that were newly generated using CRISPR-Cas systems. Third, we will identify the sulfated glycosaminoglycans bound to prion aggregates by liquid chromatography-mass spectrometry, and will determine the impact of length, abundance, and pattern of heparan sulfate chains on prion cell tropism and disease progression in mouse models. We expect that these studies will define the PrP determinants and endogenous host factors that trigger prion conversion, revealing new therapeutic targets to block prion assembly and spread.